Antagonism of endothelin actions

ABSTRACT

The mechanism of hypertension following acute NO synthase blockade is via endothelin-mediated vasoconstriction. Thus, NO appears to inhibit endothelin activity by blocking its expression and not as a chronic direct acting vasodilator. Administration of an endothelin antagonist to a patient in a ‘normal’ physiological state may result in specific regional vasodilation. This treatment finds utility in the treatment of erectile dysfunction.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a Continuation of the U.S. National Phase ofPCT/CA/00169, filed Mar. 13, 1997, which is a Continuation-in-Partapplication of U.S. Ser. No. 08/615,659, filed Mar. 13, 1996, now U.S.Pat. No. 5,688,499. The contents of these applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

[0002] This application is a continuation-in-part of U.S. Ser. No.08/615,659 filed Mar. 13, 1996, the contents of which are herebyincorporated herein by reference.

[0003] This invention relates to methods for down-regulating localendothelin-mediated vasoconstrictor and/or vascular growth activity in“apparently” normal physiological conditions in order to re-establishnormal control in specific regions of the circulation which demonstratepathophysiology. More particularly this invention relates to theadministration of agents which antagonize the expression or activity ofendothelin for the treatment of abnormalities of specific regions of thevasculature such as in erectile dysfunction in male patients.

BACKGROUND OF THE INVENTION

[0004] Endothelins were first described in 1988 and have been shown tobe powerful vasoconstrictors, predominantly found in the vascularendothelium and, since that time, numerous endothelin antagonists andpharmaceutically acceptable salts thereof have been identified and canbe obtained commercially (e.g., Sigma, American Peptides). Attention isalso directed to U.S. Pat. No. 5,284,828 issued Feb. 8, 1994 to Hemmi etal., U.S. Pat. No. 5,378,715 issued Jan. 3, 1995 to Stein et al. andU.S. Pat. No. 5,382,569 issued Jan. 17, 1995 to Cody et al., whichdescribe in detail the chemical structures of various endothelinantagonists, and to U.S. Pat. No. 5,338,726 issued Aug. 16, 1994 toShinosaki et al., which describes the chemical structure of endothelinconverting enzyme inhibitors the disclosures of which are incorporatedherein by reference. To date, however, antagonists of endothelin havenot been approved for therapeutic use, although a number ofinvestigators have postulated that endothelin antagonists could be usedfor conditions ranging from renal failure, endotoxic shock, asthma,angina, or diabetes to pulmonary hypertension and possibly otherindications.

[0005] Under normal physiological conditions, endothelin can be found inalmost all parts of the circulation at very low levels. In general, inthe normal rodent circulation endothelin (ET) is not found in elevatedquantities and appears to have minimal effect in the normal regulationof vascular tone, i.e., there is no appreciable decrease in bloodpressure when an endothelin antagonist is administered by injection innormal circulation. Further, at present there does not appear to be anyevidence suggesting that ET plays a physiological role even in a smallportion of the circulation under normal conditions in experimentalmodels. However, it is likely that the circulation may appear normalwhen in fact a specific region of the circulation revealspathophysiological changes, such as occurs with erectile dysfunction.Penile erection demands specific local vasodilation and/or inhibition oflocal vasoconstrictor mechanisms. It is not surprising that findings ofelevated levels of endothelin in the blood are not widespread, as theregulation of ET action indicates a release preferentially towards thesmooth muscle side, away from the circulation. In addition, it is highlyimprobable that there would be increased ET found in the circulationresulting from increased activity in a small portion of the circulation.ET is known to have a very short half-life.

[0006] It is widely known that administration of nitric oxide (NO) canprovoke powerful vasodilator responses. The chronic role of nitric oxidesynthase (NOS) as a vasodilator has only been inferred by indirectmeans, i.e., by removal of the NOS activity. Endogenously, there is muchmore redundancy in control of vasodilatation. For example, vasodilationcan be induced by acetylcholine, bradykinin, adenosine triphosphate(ATP), histamine, vasoactive intestinal polypeptide (VIP), andleukotrienes, amongst others. The actions of these endogenous modulatorshave been shown to be dependent on the presence of the endothelium, aneffect likely mediated by endothelial derived relaxing factor/NO(EDRF/NO) (Garg, U. C., and Hassid, A., J. Biol. Chem. 266: 9-12 (1991);Garg, U. C., and Hassid. A., J Clin. Invest. 83: 1774-1777 (1989);Palmer. R. M. J., et al., Nature 327: 524-526 (1987)). Other vasodilatormechanisms exist which are not endothelium dependent, such asβ₂-adrenergic, arial natriuretic peptide (ANP) and certainprostaglandins. The actions of NO appear to be mostly cGMP-mediated viaguanylate cyclase activation, although other mechanisms have beensuggested. Garg and Hassid (Garg, U. C. and Hassid, A., J. Biol. Chem.266: 9-12 (1991); Garg, U. C., and Hassid, A., J Clin. Invest. 83:1774-1777 (1989)) and others (Assender, J. W., et al., J. Cardiovasc.Pharmacol. 17(Suppl.3): S104-S107 (1991); O'Conner, K. J., et al., J.Cardiovasc. Pharmacol. 17(Suppl.3): S 100-S 103 (1991)) demonstrated adifference in the effects of NO-generating vasodilator agents ininhibiting vascular smooth muscle cell growth in culture; however, it isclear that NO can act not only as a vasodilator but also to inhibitvascular growth responses in a number of conditions (Farhy, R.D., etal., Circ. Res. 72: 1202-1210 (1993)).

[0007] In the last several years a large number of studies hasdemonstrated that decreased NO production using inhibitors of NOsynthase (e.g., N^(ω)-nitro-L-arginine-methyl ester or L-NAME) producesdose-dependent hypertension (i.e., L-arginine reversible, and whichcorrelates with decreased cyclic guanosine monophosphate (cGMP)) (Arnal,J. F., et al., J. Clin. Invest. 90: 647-652 (1992); Manning, R. D., Jr.,et al., J. Hypertension 21: 949-955 (1993); Manning, R. D., Jr., et al.,J. Hypertension 22: 40-48 (1993)). Data from Schiffrin's (Schiffrin, E.L., et al., J. Hypertension 25(2): 769-773 (1995); O'Conner, K. J., etal., J. Cardiovasc. Pharmacol. 17(Suppl.3): S100-S103 (1991)) andMorton's (Morton, J. J., et al., J. Hypertension, 11: 1083-1088 (1993))groups demonstrate that prolonged high dose L-NAME hypertension isassociated with hypertrophic changes in the mesenteric vasculature (↑media thickness and ↑ media/lumen ratio). Interestingly, Schiffrin'sgroup found that the degree of change in vascular structure was lessmarked than in other models (2K1C) with equivalent hypertension and of asimilar duration. Taken together with the findings of NO development ofcardiac hypertrophy and slower vascular changes, current evidenceindicates that L-NAME hypertension is quite different from other models.Further, although these findings could suggest a role for NO as amodulator of vascular structure, our recent findings suggest that NO mayplay a more important inhibitory role in suppressing the activity of theendothelin vasoconstrictor system. The concept of NO suppression of ETexpression is further supported by evidence both from Luscher's group invitro and from the Clozel group (Richard, V., et al., Circ. 91: 771-775(1995)) in vivo showing that there is increased release of ET fromendothelial cells after NOS blockage. These data suggest that exogenousadministration of NO synthase antagonists produces a condition whereinthe lack of NO appears to be a modulator of ET expression and release.Recent findings, in particular from Schiffrin's group (Schiffrin, E. L.,et al., J. Hypertension 25(2): 769-773 (1995); Schiffrin, E. L., andThibault, G., Am. J. Hypertension 4(1): 303-308 (1991)), indeoxycorticosterone acetate (DOCA)-salt hypertension point to a trophicrole for endogenous endothelin in the development of vascular structuralchanges. They found that there is increased ET-1 gene expression andimmunoreactivity in blood vessels, but not in the plasma, of DOCA-salthypertensive rats, whereas renin angiotensin system (RAS) activity wasdecreased. There was a substantial development of vascular hypertrophyin the DOCA-salt model which was markedly attenuated by treatment withan ET_(A)/ET_(B) receptor antagonist. The concept that ET-1 is avascular trophic factor is further supported by findings in studies withcultured vascular smooth muscle cells showing that addition ofendothelin produces a mitogenic response (Weber, H., et al., Mol. Endo.8(2): 148-157 (1994)), as well as findings in other in vivo studiesindicating a role in structural changes associated with pulmonaryhypertension (Eddahibi, S., et al., Am. J. Physiol. 268(2): H828-835(1995)). ET-1 is approximately 100 times more potent as avasoconstrictor than Ang II or catecholamines. Interestingly, in theculture studies, although the maximal growth response to ET-1 was lessthan half of that for Ang II, the combination of ET-1 plus Ang IIprovoked a greater mitogenic response than either peptide alone. We arenot aware of any studies that have assessed the in vivo cardiovasculargrowth responses to direct endothelin infusion.

[0008] An important aspect of the invention derives from the developmentof a concept which reveals an interrelationship between NO activity andendothelin vasoconstrictor activity, in vivo: specifically, that NO actsprimarily as a chronic inhibitor of endothelin-mediatedvasoconstriction, and less as a chronic vasodilator. Accordingly, it isproposed that endothelin plays a role in disease conditions associatedwith impaired NO synthesis, particularly if the pathophysiology isrestricted to a specific portion of the circulation; i.e., if the entirecirculation were altered, numerous compensatory changes in neurohumoralsystems would also occur.

[0009] Our hypothesis is that if NO synthesis is inhibited, asignificant increase in mean arterial pressure (MAP) is the result ofincreased endothelin release and this MAP increase can be eliminated byadministration of an endothelin antagonist. It is apparent. therefore.that administration of an endothelin antagonist in physiologicalconditions where NO production is inhibited will result in vasodilationONLY in the regions which have upregulated ET activity consequent, inpart, to a down regulation of local NO production. Physiologicalconditions where NO production is inhibited in a local circulation, suchas male erectile dysfunction, indicate that suppression of endothelinactivity would offer an effective treatment.

[0010] Based on the understanding that a significant portion of theunderlying problem in clinical erectile function relates to “vascular”mechanisms, much of the current state-of-the-art research involvesdetermining the contribution that the different vascular effectorcontrol systems make in normal and pathophysiological states. There issubstantial understanding of the hemodynamic events that lead to anerection, and yet the quantitative roles of each of the neuroeffector,humoral and local systems in these events remain poorly described. Since1990, nitric oxide (NO) has been considered the primary non-adrenergicnon-cholinergic neurotransmitter in the penis and has been presumed tobe the primary mediator of corporal relaxation during erection.

[0011] The issue of “impotence” was discussed at the National Institutesof Health (NIH) in Washington in December 1992 (defined as “a pattern ofpersistent or recurrent inability to develop or maintain an erection ofsufficient rigidity for successful coitus”) and has clearly beenidentified as having a wide range of causative or associated factors.The Massachusetts Male Aging Study (MASS) has provided us with anupdated view of the epidemiology of erectile dysfunction although thereseem to be some unchangeable truths—it is accepted that the prevalenceof impotence increases with age (Kinsey, 1948) (Kinsey, A. C., et al.,“Sexual Behavior in the Human Male”, W. B. Saunders: Philadelphia(1948)). Complete erectile dysfunction (ED) increases from 5 to 15%between 40 and 70 years of age, Feldman, 1994 (Feldman, Henry A., etal., J. Urol. 151: 54-61 (1994)). ED has been shown to be “directlycorrelated with heart disease, hypertension, diabetes, associatedmedications, indices of anger and depression, and inversely with serumdehydroepiandosterone, high density lipoprotein, cholesterol and anindex of dominant personality.”

[0012] It is now estimated that in North America there are more than30,000,000 men with ED, a significant increase from the figure of10,000,000 used just 10 years ago (Shabsigh et al., 1988 (Shabsigh, R.,et al., Urology 32: 83-90 (1988)); Whitehead, 1988 (Whitehead, E.,Geriatrics 43(2): 114 (1988)); Furlow, 1985 (Furlow, W. L., et al., Med.Aspects Human Sexuality 19: 13-16 (1985)). From these figures it is alsoreasonable to estimate that as many as three million Canadian men mayhave a degree of ED. The direct cost of treating impotence isimpressive. Reliable figures for 1985 show that the cost of treatingimpotence exceeded 146 million dollars in the United States in that yearalone (National Center for Health Statistics) and this number is justthe estimated market size for one type of injectable therapy. Thesecondary effects and indirect costs associated with erectiledysfunction would suggest that impotence and sexual dysfunction aremedical icebergs. The consequences of sexual dysfunction may be seen instrains on the host relationship potentially leading to maritalbreakdown, violence, work related sequelae, deviant sexual behavior, andimpacts on children, when present, that can carry the damage into a newgeneration of unwanted behaviors. If ED underlies even a small butsignificant percentage of marital and family breakdown, then it addsvastly to the social and economic burden in society. The pragmatic issueis that large numbers of men are now being treated for ED and most ofthe treatments are fairly blunt instruments (intracavernosal injection(ICI) of mixed vasoactive compounds, penile prosthesis insertion) withsignificant cost and complications (ICI: pain, priapism, dislike of thetechnique; prostheses: reoperation, infection, distortion of bodyimage).

[0013] As a medical and scientific problem, ED gained greatly in staturewhen Rajfer et al. (1992) (Rajfer, J., et al., New Engl. J. Med. 326(2):90-94 (1992)) published their information linking nitric oxide (NO) withnormal erectile function. It was an interesting coincidence that NObecame “Molecule of the Year” that same year as a result of theaccumulated and established work in other vascular systems. Thisheralded a new maturity in the study of ED—suddenly the principles ofnormal vascular biology (NVB) became accepted as the underpinnings oferectile physiology.

[0014] The Physiological Basis of Penile Erection

[0015] The stimulus to erection is central and neural in origin. A fullyfunctional penile erection requires coordinated input from variouslevels of the central nervous system and at least three sets ofperipheral nerves (thoracolumbar sympathetic, saccral parasympathetic,and pelvic somatic). Adrenergic, non-adrenergic and non-adrenergicnon-cholinergic neurotransmitter systems of importance have beenidentified in the cavernous tissue (Saenz de Tejada, 1988) (Saenz deTejada, I., et al., Am. J. Physiol 254: H459 (1988)). An excellentaccount of the neural processes (without specific roles) involved in theproduction of a penile erection can be found in the review by deGroatand Steers (1988) (de Groat, W. C., and Steers, W. D., “Neuroanatomy andneurophysiology of penile erection” In: Contemporary Management ofImpotence and Infertility, Tanagho, E. A., Lue, T. F., McClure, R. D.,Eds., Williams and Wilkins (1988)).

[0016] A penile erection is dependent upon the integration of anatomic,vascular (hydraulic; arterial and venous), endocrine, neurologic andhormonal mechanisms. The erectile components of the penis are thecorpora cavernosa and the corpus spongiosum. The latter contributeslittle to the rigidity of the penis when erect. The corpora cavernosaare paired cylinders that are firmly and separately anchored to theinferior pubic rami at their proximal roots, where they are covered bystriated muscle (ischiocavernosus), become joined in the proximalpendulous shaft and fenestrated (i.e., functionally connected) distally.There is usually one supplying end-artery per cavernosal body, from theinternal iliac artery, that branches to become the deep penile arterywhich has at least two types of branches within the cavernosa: Thevenous drainage of the corpora is through the intermediate system forthe distal cavernosa and glans and through the deep system for theremaining cavernosae. The critical venous channels are the subtunicalveins, which empty through emissary veins that pass through the tunicaand drain into the deep dorsal vein. It is the emissary veins that arecompressed during erection and permit the “locked” state ofveno-occlusion.

[0017] It is well established that, for erection, neurally mediated(autonomic) vasodilation of the penile arterial blood vessel and thetrabecular meshwork takes place (Lue et al., 1987) (Lue, T. F., et al,J. Urol. 137(5): 829 (1987)) permitting extra blood flow into thecavernous bodies of the penis. The expanding intracorporal volume trapsthe effluent veins that lie between the erectile tissue and thesurrounding, relatively inelastic, fibrous tunica albuginea. The outflowcapacity is thereby decreased and entrapment of blood ensues, resultingin the transformation of the flaccid penis into its erect state(Juenemann et al., 1986 (Juenemann, K. P., et al, J. Urol. 136(1): 158(1986)); Lue et al., 1987 (Lue, T. F. et al., J. Urol 137(5):829(1987)); Lue et al., 1983 (27); Weiss, 1980 (Weiss, H., et al, Ann.Intern. Med. 76: 793-799 (1980))). Inflow arterial tone is of absoluteimportance in this process, although adequate driving blood pressure(BP) is a necessary factor. The converse, detumescence, is mediated bythe sympathetic nervous system (Saenz de Tejada, 1988 (Saenz de Tejada,I., et al., Am. J. Physiol 254: H459 (1988)); Juenemann et al., 1989(Jueneman, K. P., et al., Br. J. Urol. 64-84 (1989))) and is dependenton the metabolic viability of cells within the erectile tissue. Amaximal direct pharmacological vasodilator stimulus may not produce anerection in a penis driven by the high sympathetic nervous systemactivity state induced by fear. Thus, it is not surprising thatalterations in blood flow and vascular dynamics, whether produced bydecreased cardiac output, reflex sympathetic hyperactivity,atherosclerosis, untreated hypertension, antihypertensive medication or,as herein proposed, increased local endothelin-mediatedvasoconstriction, can produce profound effects on the ability of theflaccid penis to be transformed into the erect state.

[0018] Penile Control Systems

[0019] The known control systems for erection are conventionallydescribed under the 3 headings: adrenergic, cholinergic andnon-adrenergic non-cholinergic (NANC). Adrenergic nerve fibers and highconcentrations of norepinephrine can be found in the corpora (Melman andHenry, 1979 (Melman, A., and Henry, D., J. Urol. 121: 419 (1979));Benson et al., 1980 (Benson, G. S., et al., J. Clin. Invest. 65: 506-513(1980)) and the contractile properties of phenylephrine are establishedunequivocally (Hedlund et at. 1984 (Hedlund, H. and Andersson. K. E., J.Urol, 134: 1245 (1985)); Christ et al., 1990 (Christ. G. J., et al., J.Pharmacol. 101: 375 (1990)) with post-synaptic α1 effects actingdirectly and pre-synaptic α2 modulation. Previously, parasympatheticnerves were thought to be the nerves responsible for erection (Wagner etal., 1980 (Wagner, G., and Brindley, G. S., “The effect of atrophine andc-blockers on human penile erection.” In: Vasculogenic Impotence(Zorgniotti, A. W., Rossi, G., Eds.) Charles C. Thomas: Springfield,Ill., 77-81 (1980)), although the in vitro effects from acetylcholine(ACh) were varied in early experiments (Adaikan et al., 1983 (Adaikan,P. G., et al., J. Auton. Pharm. 3: 107 (1983)); Hedlund et al., 1985(Hedlund, H., and Andersson, K. E., J. Urol., 134: 1245 (1985)).Further, simple intracorporal injection of acetylcholine does not causeerection and atropine does not block it (Wagner et al., 1980) (Wagner,G., and Brindley, G. S., “The effect of atrophine and c-blockers onhuman penile erection.” In: Vasculogenic Impotence (Zorgniotti, A. W.,Rossi, G., Eds.) Charles C. Thomas: Springfield, Ill., 77-81 (1980)).Thus, cholinergic nerves are described as modulators of neural function.Accordingly, NANC innervation, as the pre-eminent player inerectogenesis, has received intense scrutiny and the current thinking isthat nitric oxide has replaced VIP as the prime vasodilator of thissystem in the penis. This view was first published by Ignarro et al.(Ignaro, L. J., et al., Biochem. Biophys. Res. Commun. 170: 843 (1990)and has been re-stated many times since. Not surprisingly, a variety ofother NANC systems have also been shown to play a role in erectilefunction, including vasoactive intestinal polypeptide (VIP) (Gu et al.,1983 (Gu, J., et al., J. Urol. 130: 386 (1983)) &4[and 1984??] (Gu, J.,et al., Lancet2: 315 (1984)); Willis et al., 1983 (Willis, E., et al.,Life. Sci. 33: 383 (1983)), calcitonin gene related peptide (CGRP)(Stief, 1990) (Steif, C. G., et al., J. Urol. 143(2): 392-397 (1990))and the prostaglandins (Hedlund and Andersson, 1985) (Hedlund, H., andAndersson, K. E., J. Urol. 134: 1245 (1985)). The terms that have beenused to describe the neuroeffector systems and the roles they playprovide an historic basis for descriptions of penile systems but havenot removed the confusion that is found in the more than 100 relevantpapers that have been published since 1990 on neural regulation. It iswithout doubt that penile erections occur when arterial dilation andsmooth muscle relaxation take place. The penis is an ideal vascular bedto consider in terms of the physiological opposition of neural effectorsystems involved in both relaxation and contraction i.e the penis is oneof only a small handful of special circulations with dualvasoconstrictor and vasodilator neural control systems. To fullycharacterize the penile control systems, a greater understanding of thecountervailing systems both in a clinical and an experimental setting isrequired in order to elucidate the critical balance and interdependencethat are essential for normal function.

[0020] As described, in order for penile tumescence to occur, thepudendal vascular bed must vasodilate to shunt blood flow to thecavernosal tissue. Normal vascular beds have a balance of vasodilatorsand vasoconstrictors regulating the level of vascular tone. Upsetting ofthis balance can lead to an enhanced chronic vasoconstrictor response.Chronic erectile dysfunction creates a situation where the penilevascular bed has seen chronic low oxygen partial pressures pO₂. Low pO₂has been shown to decrease the activity of NO synthase and hence NOproduction. Further, it has also been shown, in rats, that the activityof the NO synthase enzyme decays with age. Both of these concepts incombination with our novel findings indicate a key role for enhancedendothelin-mediated vasoconstriction. Once enhanced endothelin occurs,there are three levels of mechanisms that will sustain the erectiledysfunction: (i) enhanced vasoconstriction in the penile vascular bedoccurs. making it more ‘difficult’ for the vasodilators to shunt bloodto the penis to facilitate cavernosal filling (this is also a positivefeedback loop with respect to NO synthase since less blood flow willmaintain low pO₂ values); (ii) endothelin has been shown in vitro and invivo to promote cardiovascular growth processes. This could lead to astructural change where blood vessels grow and encroach on the lumen,leading to increased resistance due to a structural mechanism (asopposed to chronic vasoconstriction); and (iii) enhanced endothelin mayact as a ‘primer’ for other vasoconstrictor systems (renin-angiotensinsystem and sympathetic nervous system) which additionally act as trophicfactors (i.e., the endothelin may prime the vascular bed such that AngII, for example, will promote growth at doses that by themselves wouldnot normally induce growth processes).

[0021] In summary, an upregulation of endothelin actions occurs when theproduction of NO is inhibited. This chronic enhanced endothelin, wepropose, will be involved in mediating the changes leading to erectiledysfunction. Acutely, there will be enhanced vasoconstriction via theendothelium (endothelin) and, in the longer term, endothelin-mediatedgrowth responses in the vascular tissue. The penile vascular tissuewould, therefore, go through a structural change such that it wouldbecome more and more difficult to cause vasodilation with theprogression of encroachment into the lumen of the vessels leading to thepenis as well as in the corpus cavernosal tissue itself.

[0022] There are several approaches that lead to the down regulation ofthe activity of endothelin, namely (a) peptide antagonists such asPD145065 (Parke Davis), (b) non-peptide antagonists such as bosentan(Hoffman-LaRoche) (Li, J. -S., and Schiffrin E. L., J. Hypertension13(6): 647-652 (1995)), (c) inhibitors of endothelin converting enzymesuch as for example phosphoramidon (i.e., blocking production ofendothelin) and (d) antisense oligonucleotides which specifically blockthe translation of the endothelin protein at the genetic level, i.e.,disrupt the normal cycle of events with preproendothelin mRNA.

BRIEF SUMMARY OF THE INVENTION

[0023] Thus, it is an object of the present invention to provide amethod for treating physiological conditions in which NO production isat least partially inhibited, such as, but not limited to, erectiledysfunction (ED).

[0024] Another object of this invention is to provide compositions ofmatter for the treatment of physiological conditions in which NOproduction is at least partially inhibited.

[0025] By one aspect of this invention, there is provided a method fortreating physiological conditions in which NO production is at leastpartially inhibited, comprising administering to a patient in needthereof an effective amount of an agent which will antagonize theactions of endothelin (antisense to ET-mRNA, or ET antagonists, ECEantagonists).

[0026] By another aspect of this invention, there is provided acomposition for use in the treatment of physiological conditions inwhich NO production is at least partially inhibited, comprising aneffective amount of an endothelin antagonist or pharmaceuticallyacceptable salt thereof in admixture with a pharmaceutically acceptablecarrier therefor.

[0027] In a preferred aspect, said physiological condition is erectiledisfunction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a block diagram illustrating MAP response withvasoconstrictor blockade (n=8, 8);

[0029]FIG. 2a is a graph illustrating MAP response with L-NAMEpretreated with losartan or PD 145065, (n =6, 6);

[0030]FIG. 2b is a graph illustrating MAP response after treatment withL-NAME followed by PD145065;

[0031]FIG. 3 is a graph illustrating the cumulative dose response tomethoxamine, n=6; and

[0032]FIG. 4 is a graph illustrating the cumulative dose response toendothelin, n=4.

DETAILED DESCRIPTION OF THE INVENTION

[0033] It is known that, in vivo, several systems contribute to vasculartone and mean arterial pressure (MAP). These systems include thesympathetic nervous system (SNS), renin-angiotension system and thevasopressin system. As described in more detail hereinafter, thecontribution of each of these different systems to an experimentallyinduced pressure increase of 30-40% in rats as a result of NO synthase(NOS) inhibition with N^(ω)-nitro-L-arginine-methyl ester (L-NAME, SigmaChemicals) has been assessed. If NO production is inhibited, asignificant increase in MAP must be the result of endothelin release,since this MAP increase is eliminated when an endothelin antagonist isadded. Thus, it has been established that NO acts to chronically(locally) down-regulate the release of endothelin, not as a chronicdirect acting vasodilator. The contribution of endothelin inducedvasoconstriction to L-NAME hypertension was assessed by administering anET_(A)/ET_(B) receptor antagonist such as PD 145065 (Parke DavisPharmaceuticals) in a pharmaceutically acceptable carrier therefor, bothbefore and after NO synthase inhibitor “inhibition”. Suitable carriersinclude water and isotonic dimethyl sulfoxide (DMSO). PD145065 is apeptide and hence not readily bioavailable by oral administration.Non-peptide receptor antagonists would be better choices for oraladministration. PD145065 may be effectively administered by intravenousadministration.

[0034] It will be appreciated that, in the experiments described below,an ET_(A)/ET_(B) receptor antagonist was used in order to control forany transient vasodilation which might occur via an ET_(B) receptor, andwhich would skew the estimation of involvement in hypertension followingNO synthase blockade. It is believed, however, that ET_(A) or ET_(B)receptor antagonists alone would also, at least in part, suppress theendothelin induced vasoconstriction.

[0035] ET_(A) and ET_(B) antagonists are available commercially fromvarious sources such as American Peptide Company Inc. and include: NAMEFORMULA CAT NO. ET_(A) Endothelin Antagonist c(DTrp-DAsp-Pro-DVal-Leu)88-2-10 Endothelin Receptor c(DG1u-Ala-ALLo-DIle-Leu-DTrp) 88-2-20Antagonist (BE18257B) Endothelin Antagonist (JKC-301)c(DIle-Leu-DTrp-DAsp-Pro) 88-2-30 Endothelin Antagonist (JKC-302)c(DVaL-Leu-DTrp-DSer-Pro) 88-2-31 Endothelin Antagonist (BQ-610)(N,N-hexamethylene)carbamoyl-Leu- 88-2-32 DTrp(CHO)-DTrp EndothelinReceptor Antagonist c(DGLu-Ala-DVal-Leu-DTrp) ET_(B) [Cys 11, Cys15]Endothelin-1 c(DGlu-Ala-DVal-Leu-DTrp) 88-2-41 (8-21), (1RL-1038)[Ala 11,15]Endothelin-1 (6-21), Ac-Leu-Met-Asp-Lys-Glu-Ala-Tyr-Phe-88-2-42 N-Acetyl Ala-His-Leu-Asp-Ile-Ile-Trp N-Suc-[Glu9,Ala11,15]Endothelin- Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe- 88-2-50 1, (8-21),(LRL-1620) Ala-His-Leu-Asp-Ile-Ile-Trp

[0036] Methods

[0037] Animals

[0038] Male Sprague-Dawley rats (325-400 g) obtained from Charles RiverLaboratories (Montreal, Quebec, Canada) were housed individually underconditions of a 12 h light/12 h dark cycle, with room temperature at22-24↑C, and were provided with Purina Rodent Chow and tap water adlibitum for at least 2 days before any experiments were started.

[0039] Measurement of MAP and Short Acting Drug Administration

[0040] The surgical method was based on the technique of Thompson et al.(Hypertension 20: 809-815 (1992)). In brief, rats were anesthetized withketamine/xylazine (70/5 mg/kg i.p.), and the descending aorta distal tothe kidneys was catheterized with small bore TEFLON® tubing (0.012-in.i.d., o.d. 30 gauge, Cole-Parmer, Laval, Quebec, Canada) inserted intovinyl tubing (0.02-in. i.d., 0.060-in. o.d., 23 gauge). The inferiorvena cava was also catheterized distal to the kidneys with small boreTEFLON® tubing (0.012-in. i.d., o.d. 30 gauge, Cole Parmer). Thecatheters were filled with heparinized saline (10 IU/mL) and held inplace by a small amount of cyanoacrylate tissue glue at the puncturesite. The catheters were tunneled subcutaneously and exteriorized at theback of the neck and sutured in place. Two days after surgery, MAP couldbe recorded (Narco Physiograph, E&I Instruments, Houston, Tex. or MacLabData Acquisition System, ADInstruments, Milford, Mass., U.S.A.). Afterconnection, an equilibration period of approximately 30 min was allowedfor the determination of the steady state level of MAP before anyrecording began. Baseline MAP was determined from mean readings over 5min, taken from each rat at 15 min intervals for at least 1 h prior tothe start of any experiment. After obtaining a baseline MAP, thecumulative/sequential vasoconstrictor blockade was started. MAP valuesare expressed as an average MAP over 2-3 min following the establishmentof a new steady state (i.e., MAP is no longer changing).

EXPERIMENT 1 Cumulative Vasoconstrictor Blockade Following Acute NOInhibition

[0041] NOS was inhibited by a single i.p. injection ofN^(ω)-nitro-L-arginine-methyl ester (L-NAME, Sigma, 100 mg/kg; 100 mg/mL0.9% sterile saline solution, Baxter Corp., Toronto, Ontario, Canada).The α₁-adrenoceptor antagonist prazosin® (Sigma Chemical Company, St.Louis, Mo. U.S.A.) at a dose of 1 mg/kg (in 0.9% saline and 5% ethanol[vol/vol], total volume 1 mL/kg) was used to block the majority of theSNS induced peripheral vasoconstriction. The non-peptide AT₁ receptorantagonist losartan® (Dupont-Merck, 30 mg/kg i.p.) was used to block theeffects of Ang II. The V₁/V₂ receptor antagonist [β-mercapto-β,β-cyclopenta-methylenepropionyl, O-Et-Tyr₂-Val₄-Arg₈]-vasopressin (AVP)(20 μg/kg/min i.v., Sigma) was utilized to block the effects ofvasopressin. The effects of the endothelin vasoconstrictor system, viaET_(A) and ET_(B) receptors, were blocked with a commercially availableendothelin antagonist PD 145065 (100 mg/kg/min i.v., bolus, donated byParke-Davis Pharmaceuticals). Each rat in the study received all of theaforementioned vasoconstrictor antagonists (i.e., acumulative/sequential blockade). The addition of sodium nitroprusside(SNP) (200 μg/kg i.v. 0.3 c.c. bolus, Sigma) was used to transientlylower MAP to the level of minimum vascular resistance across alltreatment groups (i.e., where the level of MAP becomes predominantlydependent on cardiac output at minimum vascular resistance;MAP_(min)=CO×TPR_(min)). Not surprisingly, this value was found to besimilar across all treatment groups and enabled the calculation of thetotal range of MAP pressure lowering (MAP_(range)=MAP−MAP_(min)). Aswell, the addition of SNP reveals the activity of ‘other’vasoconstrictor systems that might not have been accounted for but maybe important in contributing to the level of MAP in control rats orafter acute L-NAME treatment.

[0042] The selective blockers of the various vasoconstrictor systemswere added in a cumulative and sequential manner in the following order:prazosin losartan, AVP antagonist, PD 145065 and SNP. The intervalbetween administration was up to 15 min, where a new steady state MAPhad been established before the blockade of the next vasoconstrictorsystem.

EXPERIMENT2 Effects of PD 145065 Before or After NOS Blockade

[0043] After obtaining appropriate baseline steady-state levels of MAP,the effects of administration of losartan (30 mg/kg i.p. n=3, under 30mg/kg i.p. pentabarbital anesthetic) or PD 145065 (10 mg/kg/min, i.v.,n=6, conscious rats previously catheterized) on resting MAP and then onthe L-NAME (100 mg/kg, i.p.)-induced pressor response were assessed intwo ways. Administration of PD145065 was done prior to giving L-NAME toassess the effect on the normal circulation as well as on thedevelopment of the hypertension, whereas the administration afterL-NAME-hypertension was already present was used to assess reversibilityof the pressor response.

[0044] Data Analysis

[0045] All data are expressed as a peak MAP lowering as a mean±S.D.Comparison of means between groups at each treatment level was doneusing a Student's T-test with the Bonferroni correction method, whereappropriate.

[0046] Results

[0047]FIG. 1 illustrates the changes in steady state MAP responsesfollowing the sequential blockade of the vasoconstrictor systems in boththe L-NAME (n=6) and saline control (n=6) groups. The steady statelevels of MAP for saline and L-NAME-treated animals were 146±14 and108±9 mmHg, respectively. The administration of large doses ofprazosin®, losartan® and (O-ET) VAVP to antagonize the effects of 3 ofthe major vasoconstrictor systems lowered MAP by a similar extent inboth the L-NAME (84±13 mmHg)-and saline (46±5 mmHg)-treated groups bothon an absolute (delta MAP) and proportional (% of range) basis (Table 1below) and yet there remained the same pressor response in the L-NAMEgroup compared to control. TABLE 1 Saline L-NAME Blocking agent deltaMAP % of range delta MAP % of range prazosin 36 ± 6 48 ± 08 27 ± 14* 25± 10* losartan 20 ± 6 27 ± 8 25 ± 13 24 ± 12 (O-ET)VAVP 21 ± 11 37 ± 1519 ± 13 15 ± 9* PD 145065  1 ± 2  1 ± 3 35 ± 13* 39 ± 24* SNP  7 ± 3 12± 4 12 ± 9  4 ± 2

[0048] The addition of PD 145065 to the saline-treated group followingprazosin, losartan and (O-ET) VAVP administration did not significantlylower further the level of MAP (46±4 mmHg). In contrast, the addition ofPD 145065 to the L-NAME-treated group under similar conditionsdramatically lowered MAP from 84±13 mmHg to 46±7 mmHg. Theadministration of SNP further lowered MAP to 39±3 mmHg across alltreatment groups, revealing a small component of other systems in bothtreatment groups.

[0049] In Experiment 2, after obtaining appropriate baseline levels ofMAP (100±15 mmHg), losartan treatment resulted in a lowering of MAP to78±21 mmHg. In contrast, the administration of PD 145065 had no effecton the level of MAP (106±16 mmHg). There was a marked difference in theL-NAME pressure profile between the saline-and PD 145065-treated group(FIG. 2). The delta MAP responses 20 min after L-NAME treatment in thesaline and PD 145065 pre-treatment groups were 40±8 and 11±7 mmHg,respectively (p<0.05).

[0050] The above experiments demonstrate that the contribution of thesympathetic nervous system, renin-angiotensin system and vasopressinsystem are not enhanced in the acute L-NAME induced hypertensive state,yet there remains a markedly enhanced vasoconstrictor tone (=↑40%)compared to control. These studies also demonstrate that the pre-eminentmechanism of hypertension following acute NO synthase blockade is viaendothelin-mediated vasoconstriction. This endothelin mechanism suggeststhat the prominent role of NO is to inhibit endothelin activity, likelyby inhibiting its expression and release and not as a chronic directacting vasodilator. Experiment 2 shows that pharmacological antagonismof the endothelin receptors does not change resting MAP, confirmingprevious studies that endothelin does not function as a prominentvasoconstrictor in normal circulating states.

[0051] There are, however, certain conditions which may be consideredphysiologically normal but in which local levels of endothelin causevasoconstriction, such as erectile responses in males. Administration ofan A/B endothelin receptor antagonist in such circumstances will cause adesired local vasodilation and thus permit penile erection in anotherwise impotent male, and will decrease the potential for long termchanges in vascular function and structure.

[0052] In situ Experimental Evidence for Endothelin as a ‘Primer’

[0053] Three points regarding the mechanisms of endothelin involvementin erectile dysfunction have been described hereinabove. There is nowpresented in situ evidence to demonstrate that enhanced levels ofendothelin markedly ‘sensitize’ the pudendal vascular bed to othervasoconstrictor systems. Stated another way, a slight enhancement ofendothelin will synergistically enhance the impact of the sympatheticnervous system, i.e., a marked increase in vasoconstriction will resultfrom even normal levels of sympathetic activation.

[0054] Vascular responses using the isolated perfusion of the pudendalvasculature preparation have been assessed. FIG. 3 illustrates the doseresponse curve to the α-adrenoceptor agonist methoxamine (MXA) alone.FIG. 4 illustrates the dose-response curve to endothelin alone. Table 2represents the changes in perfusion pressure when low, sub-pressor dosesof endothelin+MXA are given in comparision to MXA alone. These findingsdemonstrate that the MXA concentration-response (perfusion pressure)relationship was shifted to lower concentrations by more than 10-fold,i.e., when low doses of endothelin and MXA which alone are sub-pressorwere combined, the effect was a pronounced increase in vascularresistance. These findings demonstrate that low level endothelinstimulation in the penile vasculature will result in synergism whena-adrenergic receptors are activated.

[0055] Methods

[0056] Animals

[0057] Male Wistar rats (400-500 g), obtained from Charles RiverLaboratories (Montreal, Quebec, Canada), were housed individually underconditions of a 12 h light/12 h dark cycle (temperature of 22-24° C.),and were provided with Purina Rodent Chow and tap water ad libitum forat least two days before starting any experiment.

[0058] Pudendal Perfusion Preparation

[0059] The rats were anaesthetized with sodium pentabarbital (60 mg/kgi.p.) and the lower abdominal aorta was exposed through a mid-lineincision. The isolation of the pudendal vasculature was developed bysequential ligation of all branches of the abdominal aorta not directlysupplying the pudendal vasculature. The following arteries weredissected free and ligated: iliaca. femoralis, poplitea, glutea cranial,umbilicalis, epigastria caudal, pudenda external, glutea caudal,obturatoria, circumflexa femoris lateral, circumflexa femoris medial.After heparinization (1000 U/kg), the abdominal aorta was cannulatedwith a smooth, blunted 19-gauge needle. The arterial catheter was placedcaudad into the aorta and the vena cava was cut and vented freelythrough the widely open abdominal cavity. The tip of the needle wasadvanced down the aorta to the iliac bifurcation and sutured in place.Flow of perfusate (0.5 mL/kg body weight/min) through the abdominalaorta was started immediately after transection, with sharp surgicalscissors of the abdominal aorta, inferior vena cava spinal cord and alltissue 1 cm lateral to the spinal cord between T₃ and T₇ (FIG. 2). Theperfusate was infused for 10 min, to flush the penile vasculature ofblood, before starting any experiment.

[0060] The perfusate consisted of dextran (15%, average molecularweight: 71,400 Da, Sigma, St. Louis, Mo., U.S.A.) in Tyrode's solution(pH 7.4), which was aerated with 95% O₂ and 5% CO₂ The composition ofthe Tyrode's solution was KCl20, CaCl₂↑2H₂0 32.3, MgCl₂↑6H₂O 5.1,NaH₂PO₄↑2H₂O 6.2, NaHCO₃ 100, glucose 100, and NaCl 800 mg per 100 mLfluid. The perfusate was held in a reservoir, and passed through abubble trapping/mixing chamber and heating bath by an injection portlocated prior to the bubble trap for the introduction of pharmacologicalagents. An in-line peristaltic pump was used to establish flow at 0.5mL/kg/min (Minipuls 2, Gilson Medical Elec., Inc., Middleton, Wis.,U.S.A.). Added pharmacological agents, methoxamine (MXA) and sodiumnitroprusside (SNP), were delivered by a syringe pump (Harvard ApparatusInfusion/Withdrawal Pump, Millis, Mass., U.S.A.). A servo-controlledheat chamber served to maintain rectal temperature at 36-38° C. TABLE 2A comparision of the perfusion pressure responses in control and endo-thelin subpressor treated rats to the α₁-adrenoceptor agonistmethoxamine. (Δ Perfusion Pressure (mmHg)) [methoxamine μg/ml ControlEndothelin sub-pressor 0  0  0 0.1  0  0 0.25  0  34 ± 2 0.5  0  93 ± 301.0  4 ± 1 168 ± 16 2.0 11 ± 2 — 4.0 36 ± 11 — 8.0 67 ± 24 —

[0061] Values are expressed as group mean±S.D., n=6 for control, n=4 forendothelin sub-pressor.

EXPERIMENT Effects of Endothelin Antagonists on Blockade ofApomorphine-induced Erections

[0062] It is well known that NOS blockers (e.g., L-NAME) inhibiterectile response acutely. According to our novel understanding ofnormal vascular biology as described herein, we proposed that themechanism of L-NAME-induced erectile dysfunction, at least acutely, isdependent on the activation of endothelin (ET)-mediatedvasoconstriction, and can thus be reversed with an ET antagonist. Thiswas confirmed experimentally as described below.

[0063] Methods

[0064] Using the well-established apomorphine (APO)-induced erectionmodel (apomorphine 80 μg/kg, administered subcutaneously), we countedthe erections per 30 min interval in adult Wistar rats, per the protocolof Heaton, J. P. W., et al. (1991)( Heaton, J. P. W., et al., J. Urol.145: 1099-1102 (1991)). Rats were pretested with apomorphine alone toconfirm and quantify normal erectile response(control Groups A and B).Rats were then treated with L-NAME (100 mg/kg, administeredintraperitoneally) alone, acutely (at <1 h) and at 3 h; or thecombination of ET_(A/B) antagonist (PD 145065, 5 mg/kg/min, continuousintravenous infusion)+L-NAME.

[0065] Results

[0066] Data represent mean number of erections±S.D. in the variousdifferent treatment groups. L-NAME alone blocked erection. Addition ofthe ET antagonist completely reversed acute L-NAME-induced decrease inerections, resulting in normal incidence of apomorphine-inducederections. Group-A Group-B PD 145065 + (control) (control) L-NAME-acuteL-NAME L-NAME-3 h 3.0 ± 1.31 2.5 ± 0.58 1.4 ± 0.74** 2.5 ± 0.58 3.7 ±1.38

CONCLUSIONS

[0067] The mechanism of decreased erectile function following acuteL-NAME involves increased endothelin-mediated vasoconstriction.

[0068] By this experiment in combination with the foregoing, theinventors provide proof of concept of their novel model of normalvascular biology and of the use of endothelin antagonists in downregulating local endothelin-mediated vasoconstrictor tone and vasculargrowth activity in a patient independently of any normal or abnormalsystemic physiology.

[0069] All scientific publications cited herein are hereby incorporatedby reference.

[0070] Although this invention is described in detail with reference topreferred embodiments thereof, these embodiments, like the experimentsdescribed herein, are offered to illustrate but not to limit theinvention. It is possible to make other embodiments that employ theprinciples of the invention and that fall within its spirit and scope asdefined by the claims appended hereto.

What is claimed is:
 1. A method for treating physiological conditions inwhich NO production is at least partially inhibited, comprising the stepof administering to a patient in need thereof an effective amount of anagent which will antagonize the actions of endothelin, in apharmaceutically acceptable carrier therefor.
 2. A method as claimed inclaim 1 wherein said agent is selected from the group consisting ofpeptidal endothelin antagonists, non-peptidal endothelin antagonists,inhibitors of endothelin converting enzyme, and antisenseoligonucleotides which block translation of endothelin mRNA.
 3. A methodas claimed in claim 2 wherein said peptidal endothelin antagonist is anET_(A)/ET_(B) receptor antagonist.
 4. A method as claimed in claim 2wherein said non-peptidal endothelin antagonist is bosentan.
 5. A methodas claimed in claim 2 wherein said inhibitor of endothelin convertingenzyme is phosphoramidon.
 6. A method as claimed in claim 2 wherein saidanti-sense oligonucleotide is complementary to at least a portion ofpreproendothelin mRNA.
 7. A method as claimed in claim 1 wherein saidphysiological condition is selected from the group consisting ofhypertension and erectile dysfunction.
 8. A method as claimed in claim 2wherein said agent is administered orally.
 9. A method as claimed inclaim 2 wherein said agent is administered intraperitoneally.
 10. Amethod as claimed in claim 3 wherein said ET_(A)/ET_(B) receptorantagonist is PD145065.
 11. A method as claimed in claim 10 wherein saidPD145065 is administered intraperitoneally.
 12. A composition for use inthe treatment of physiological conditions in which NO production is atleast partially inhibited, comprising an effective amount of an agentwhich will antagonize the action of endothelin, in admixture with apharmaceutically acceptable carrier therefor.
 13. A composition asclaimed in claim 12 wherein said agent is selected from the groupconsisting of peptidal endothelin antagonists, non-peptidal endothelinantagonists, inhibitors of endothelin converting enzyme and antisenseoligonucleotides which block translation of endothelin mRNA.
 14. Acomposition as claimed in claim 13 wherein said peptidal endothelinantagonist is an ET_(A)/ET_(B) receptor antagonist.
 15. A composition asclaimed in claim 14 wherein said ET_(A)/ET_(B) receptor antagonist isPD145065.
 16. A composition as claimed in claim 13 wherein saidnon-peptidal endothelin antagonist is bosentan.
 17. A composition asclaimed in claim 13 wherein said inhibitor of endothelin convertingenzyme is phosphoramidon.
 18. A composition as claimed in claim 13wherein said antisense oligonucleotide is complementary to at least aportion of preproendothelin mRNA.
 19. A composition as claimed in claim13 wherein said agent is an ET_(A) receptor antagonist.
 20. Acomposition as claimed in claim 13 wherein said agent is an ET_(B)receptor antagonist.
 21. A method for down regulating localendothelin-mediated vasoconstrictor tone and vascular growth activity ina patient independently of any normal or abnormal systemic physiology,comprising the step of administering an effective amount of endothelinantagonist agent in a pharmaceutically acceptable carrier therefor. 22.A method as claimed in claim 21 wherein said agent is selected from thegroup consisting of peptidal endothelin antagonists, non-peptidalendothelin antagonists, inhibitors of endothelin converting enzyme, andantisense oligonucleotides which block translation of endothelin mRNA.23. A method as claimed in claim 22 wherein said peptidal endothelinantagonist is an ET_(A)/ET_(B) receptor antagonist.
 24. A method asclaimed in claim 21 wherein said vasoconstrictor tone is selected fromthe set consisting of the tone of the pudendal vasculature, the tone ofthe arteries feeding the pudendal vasculature, and a combinationthereof.
 25. A composition for use in down regulation of localendothelin-mediated vasoconstrictor tone and vascular growth activity ina patient independently of any normal or abnormal systemic physiology,comprising an effective amount of an agent which will antagonize theaction of endothelin, in admixture with a pharmaceutically acceptablecarrier therefor.
 26. A composition as claimed in claim 25 wherein saidagent is selected from the group consisting of peptidal endothelinantagonists, non-peptidal endothelin antagonists, inhibitors ofendothelin converting enzyme and antisense oligonucleotides which blocktranslation of endothelin mRNA.
 27. A composition as claimed in claim 26wherein said peptidal endothelin antagonist is an ET_(A)/ET_(B) receptorantagonist.
 28. A composition as claimed in claim 25 wherein saidvasoconstrictor tone is selected from the set consisting of the tone ofthe pudendal vasculature, the tone of the arteries feeding the pudendalvasculature, and a combination thereof.